Ezh2 knockout and gene expression profiling in CD8+ T cells. Mus musculus

We used 6 male 4 week old C57BL/6 (B6) mice, three with wild-type (WT) genes, the other 3 with conditional knockout (KO) of both Ezh2 alleles. Knockout was achieved by breading B6 mice with floxed alleles of Ezh2 (Ezh2-fl/fl) to B6 mice expressing Cre recombinase under the control of a CD4 promotor to generate T-cell specific homozygous Ezh2 conditional knockout (CKO) mice. CD8 T-cells were purified using anti-mouse CD8 antibody (Ab)-conjugated magnetic beads from the spleen and lymph nodes of WT and CKO mice. The purity was consistently more that 93% as assessed by flow cytometry analysis. Freshly isolated mature T cells were named naïve CD8 T cells. These naïve CD8 T cells (two million cells / ml) were cultured in media containing 10% fetal bovine serum-IMDM in the presence of plate-coated anti-CD3 Ab (3 mg /ml) + anti-CD28 Ab (3 mg /ml). Three days later, these in vitro stimulated CD8 T cells were collected and named activated CD8 T cells. About 200 ng total RNA was extracted, and amplified using small-sample methods to make biotin-labeled cDNA targets that were hybridized to Affymetrix HT Mouse 430 PM arrays. This resulted in 4 groups of samples: naive or activated cells that were either KO or WT for Ezh2, each group having three biological replicates. The samples are in theory paired, since pairs of samples came from the same mouse, however we did not find many significant mouse to mouse effects, and do not think they need to be modeled. We provide a supplementary Excel workbook that holds the expression data, but it also holds probe-set annotation, and some statistical calculations (and the functions used to compute them), and may be convenient for some users. As of Dec 20, 2018, we were not expecting to use this data in a publication in the near future, and so released it to the public. Overall design: We used 6 male 4 week old C57BL/6 (B6) mice, three with wild-type (WT) genes, the other 3 with conditional knockout (KO) of both Ezh2 alleles. Knockout was achieved by breading B6 mice with floxed alleles of Ezh2 (Ezh2-fl/fl) to B6 mice expressing Cre recombinase under the control of a CD4 promotor to generate T-cell specific homozygous Ezh2 conditional knockout (CKO) mice. CD8 T cells were purified using anti-mouse CD8 antibody (Ab)-conjugated magnetic beads from the spleen and lymph nodes of WT and CKO mice. The purity was consistently more that 93% as assessed by flow cytometry analysis. Freshly isolated mature T cells were named naïve CD8 T cells. These naïve CD8 T cells were cultured in media containing 10% fetal bovine serum-IMDM in the presence of plate-coated anti-CD3 Ab (3 mg /ml) + anti-CD28 Ab (3 mg /ml). Three days later, these in vitro stimulated CD8 T cells were collected and named activated CD8 T cells. About 200 ng total RNA was extracted, and amplified using small-sample methods to make biotin-labeled cDNA targets that were hybridized to Affymetrix HT Mouse 430 PM arrays. This resulted in 4 groups of samples: naive or activated cells that were either KO or WT for Ezh2, each group having three biological replicates.

本研究使用6只4周龄雄性C57BL/6（B6）小鼠，其中3只为野生型（wild-type, WT）基因小鼠，剩余3只为Ezh2等位基因双条件性敲除（conditional knockout, KO）小鼠。条件性敲除的构建方式为：将携带Ezh2 flox等位基因（Ezh2-fl/fl）的B6小鼠，与在CD4启动子调控下表达Cre重组酶的B6小鼠交配，从而获得T细胞特异性纯合Ezh2条件性敲除（CKO）小鼠。采用抗小鼠CD8抗体（Ab）偶联磁珠，从野生型与CKO小鼠的脾脏及淋巴结中纯化CD8 T细胞；经流式细胞术（flow cytometry）检测，细胞纯度始终高于93%。新鲜分离的成熟T细胞被命名为初始CD8 T细胞（naïve CD8 T cells）。将上述初始CD8 T细胞以2×10^6个/mL的密度接种于含10%胎牛血清的IMDM培养基中，并加入包被于培养板的抗CD3 Ab（3 mg/mL）与抗CD28 Ab（3 mg/mL）进行刺激培养。培养3天后，收集经体外刺激的CD8 T细胞，命名为活化CD8 T细胞（activated CD8 T cells）。提取约200 ng总RNA，采用微量样本扩增方法制备生物素标记的cDNA靶标，随后与Affymetrix HT Mouse 430 PM基因芯片进行杂交。最终得到4组样本：分别为Ezh2基因敲除或野生型的初始细胞、活化细胞，每组均设置3次生物学重复。理论上样本为配对样本，即每对样本取自同一只小鼠，但本研究未观察到显著的个体间效应，因此认为无需进行配对建模。本研究附带一份补充Excel工作簿，其中包含表达谱数据、探针集注释信息、部分统计计算方法（含计算所用函数），可为部分使用者提供便利。截至2018年12月20日，本研究暂无近期将该数据用于学术论文发表的计划，故将其公开发布。实验整体设计如下：本研究使用6只4周龄雄性C57BL/6（B6）小鼠，其中3只为野生型（wild-type, WT）基因小鼠，剩余3只为Ezh2等位基因双条件性敲除（conditional knockout, KO）小鼠。条件性敲除的构建方式为：将携带Ezh2 flox等位基因（Ezh2-fl/fl）的B6小鼠，与在CD4启动子调控下表达Cre重组酶的B6小鼠交配，从而获得T细胞特异性纯合Ezh2条件性敲除（CKO）小鼠。采用抗小鼠CD8抗体（Ab）偶联磁珠，从野生型与CKO小鼠的脾脏及淋巴结中纯化CD8 T细胞；经流式细胞术（flow cytometry）检测，细胞纯度始终高于93%。新鲜分离的成熟T细胞被命名为初始CD8 T细胞（naïve CD8 T cells）。将上述初始CD8 T细胞以2×10^6个/mL的密度接种于含10%胎牛血清的IMDM培养基中，并加入包被于培养板的抗CD3 Ab（3 mg/mL）与抗CD28 Ab（3 mg/mL）进行刺激培养。培养3天后，收集经体外刺激的CD8 T细胞，命名为活化CD8 T细胞（activated CD8 T cells）。提取约200 ng总RNA，采用微量样本扩增方法制备生物素标记的cDNA靶标，随后与Affymetrix HT Mouse 430 PM基因芯片进行杂交。最终得到4组样本：分别为Ezh2基因敲除或野生型的初始细胞、活化细胞，每组均设置3次生物学重复。